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. 2021 Sep 21;11:690618. doi: 10.3389/fonc.2021.690618

Figure 4.

Figure 4

Latent TGFβ1 can bind to ITGB6 and release active TGFβ1 to stimulate the Smad3 pathway. (A) A dual-luciferase reporter assay showed changes in 3D-cultured HEY cells after ITGB6 silencing. (B) Changes in the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after treatment with or without latent TGFβ1 and anti-ITGB6 antibody. Anti-ITGB6 antibody (10 μg/ml) was added to the culture medium as a pretreatment to block ITGB6 for 12 h. Then, 10 ng/ml latent TGFβ1 was added to the culture medium for 72 h (equal doses of normal mouse IgG1 and PBS were used as the negative controls for the anti-ITGB6 antibody and latent TGFβ1, respectively). (C) Changes in the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after silencing of ITGAV. NC-ITGAV and si-ITGAV were transfected into HEY and A2780 cells. After 6 h, the cells were resuspended in ultralow-attachment plates for 3D culture. Then, 10 μg/ml anti-ITGB6 antibody was used to block ITGB6 for 12 h in 3D-cultured NC-ITGAV and si-ITGAV cells, and equal doses of normal mouse IgG1 were used as a negative control. Subsequently, 10 ng/ml latent TGFβ1 was added to the culture medium and incubated for 72 h in each group. In the histogram of the quantification, “*” refers to p < 0.05, “**” refers to p < 0.01, and “***” refers to p < 0.001.